Journal: EBioMedicine

Article Title: Proapoptotic Cyclic Peptide BC71 Targets Cell-Surface GRP78 and Functions as an Anticancer Therapeutic in Mice

doi: 10.1016/j.ebiom.2018.06.004

Figure Lengend Snippet: BC71 targets cell-surface GRP78 but not αvβ5 integrin to induce apoptosis. (a) BC71 induces HUVECs apoptosis in a dose-dependent manner. HUVECs were treated with BC71 (concentration range: 12.5, 25, 50, 100 μM) for 24 h and apoptosis was determined using the cell death ELISA kit (Roche). (b) Anti-GRP78 N-terminal domain antibody blocked the apoptosis function of BC71 in a dose-dependent manner. The apoptosis of the combined treatment with increasing amount of anti-GRP78 N-terminal domain antibody and 100 μM BC71 for 24 h was measured using the Cell Death Detection ELISA. (c) Anti-GRP78 C-terminal domain antibody and (d) anti-αvβ5 antibody did not block BC71 induced apoptosis. For clarity of presentation, data were normalized with that of non-treated (VEGF only) cells, which was set as 1. Data are expressed as mean ± standard error of the mean. The results are representative of at least three independent experiments. Statistical significance was determined using ANOVA. *P < 0.05; **P < 0.01, n ≥ 3.

Article Snippet: Antibodies against integrin αvβ5 (P1F76; Santa Cruz Biotechnology, Santa Cruz, CA, USA), GRP78 (A-10, Santa Cruz Biotechnology) and GRP78 (C-20, Santa Cruz Biotechnology) were used for neutralization.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay

Journal: Nature communications

Article Title: Osteopontin is a therapeutic target that drives breast cancer recurrence.

doi: 10.1038/s41467-024-53023-9

Figure Lengend Snippet: Fig. 1 | β1 integrin-deficient recurrent tumors have elevated levels of osteo- pontin (OPN, Spp1). a UMAP plots showing Spp1 expression from single-cell RNA sequencing of early invasive carcinoma from MIC WT lesions (fast-growing, pooled lesions from n = 3 mice) or MIC β1KO lesions (dormant, pooled lesions from n = 6 mice), specifically in the epithelial tumor cell cluster. b Normalized read counts for Spp1 from RNA-seq data from MIC WT (n = 6) or MIC β1KO (n = 9) recurrent tumors that exited dormancy from GSE186491. c RT-qPCR analysis for Spp1 transcript, normalized to Gapdh from MIC WT (n = 6) and MIC β1KO (n = 13) recurrent tumors. d RNA Scope for mouse Spp1 (mSpp1) and fluorescent immunohistochemistry

Article Snippet: The following antibodies were used for immunoblots: Stat3 (CST, 9139, 1:1000), p-Stat3 (CST, 9145, 1:1000), β-actin (Sigma, A5441, 1:2000), tubulin (CST, 2148, 1:1000), β3 integrin (Abcam, Ab119992, 1:1000), αV integrin (CST, 60896, 1:1000) and CD44 (CST, 37259, 1:1000).

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, RNAscope, Immunohistochemistry

Journal: Nature communications

Article Title: Osteopontin is a therapeutic target that drives breast cancer recurrence.

doi: 10.1038/s41467-024-53023-9

Figure Lengend Snippet: Fig. 3 | β1 integrin-deficient recurrent tumors have elevated levels of pro- tumorigenic macrophages. a Fluorescent IHC for F4/80, CD206, and DAPI on MIC WT (n = 15) and MIC β1KO (n = 15) recurrent tumors. b, c Quantification of total F4/ 80+ cells (total macrophages) and F4/80+ CD206+ cells (pro-tumorigenic macro- phages) in MIC WT (n = 15) and MIC β1KO (n = 15) recurrent tumors. d RNA Scope for mouse IL-4 and fluorescent IHC for PanCK and DAPI on MIC WT (n = 25) and MIC β1KO (n = 18) recurrent tumors. e, f Quantification of total IL-4+ cells and IL-4+ PanCK+ cells in MIC WT (n = 25) and MIC β1KO (n = 18) recurrent tumors. g Representative MIC WT (n = 15) and MIC β1KO (n = 15) recurrent tumors from (a)

Article Snippet: The following antibodies were used for immunoblots: Stat3 (CST, 9139, 1:1000), p-Stat3 (CST, 9145, 1:1000), β-actin (Sigma, A5441, 1:2000), tubulin (CST, 2148, 1:1000), β3 integrin (Abcam, Ab119992, 1:1000), αV integrin (CST, 60896, 1:1000) and CD44 (CST, 37259, 1:1000).

Techniques: RNAscope

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Acupuncture Delays Cartilage Degeneration through Upregulating SIRT1 Expression in Rats with Osteoarthritis

doi: 10.1155/2021/2470182

Figure Lengend Snippet: Acupuncture inhibited OA-associated inflammation, the NF- κ B signaling pathway, and ECM degradation through upregulating SIRT1 expression in rat articular cartilages. (a/b): The levels of TNF- α and IL-2 in the serum of acupuncture-treated OA rats injected with or without shSIRT1 lentiviruses were assessed by enzyme-linked immunosorbent assay. (c/d/e/f/g/h): The expressions of SIRT1, MMP-9, ADAMTS5, p-p65/p65, p-I κ B α /I κ B α , collagen II, and aggrecan in the articular cartilage of acupuncture-treated OA rats injected with or without shSIRT1 lentiviruses were analyzed by western blot, with GAPHD serving as a reference gene. ∧ P or ‡ P < 0.05; ∗∗ P or ∧∧ P or ## P or ‡‡ P < 0.01; ∗∗∗ P or ^^^ P or ### P or ‡‡‡ P < 0.001; ∗ vs. Sham; ∧ vs. Model + shNC; # vs. Model + Acupuncture + shNC; ‡ vs. Model + shSIRT1 (OA: Osteoarthritis; TNF- α : Tumor necrosis factor- α ; IL-2: interleukin-2; shNC: shRNA-negative control; MMP-9: matrix metallopeptidase-9; SIRT1: NAD-dependent deacetylase sirtuin-1; ADAMTS5: a disintegrin and metalloproteinase with thrombospondin motifs 5).

Article Snippet: The membranes were blocked by 5% nonfat milk (P2194, Sigma-Aldrich, USA) in tris buffered saline with 1% Tween 20 (TBST; TA-125-TT, ThermoFisher, USA) for 1 h and further probed with primary antibodies against SIRT1 (#9475, 120 kDa, 1 : 1000, Cell Signaling Technology, Danvers, MA, USA), matrix metallopeptidase (MMP)-9 (ab76003, 92 kDa, 1 : 1000, Abcam, USA), A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5; ab41037, 73 kDa, 1 : 250, Abcam, USA), phosphorylated (p)-p65 (#3033, 62 kDa, 1 : 1000, Cell Signaling Technology, USA), p65 (#8242, 65 kDa, 1 : 1000, Cell Signaling Technology, USA), p-I κ B α (#2859, 40 kDa, 1 : 1000, Cell Signaling Technology, USA), I κ B α (#4812, 39 kDa, 1 : 1000, Cell Signaling Technology, USA), collagen II (ab188570, 141 kDa, 1 : 1000, Abcam, USA), aggrecan (orb624552, 250 kDa, 1 : 500, Biorbyt, Cambridge, UK), and GAPDH (ab8245, 36 kDa, 1 : 1000, Abcam, USA) at 4°C overnight.

Techniques: Expressing, Injection, Enzyme-linked Immunosorbent Assay, Western Blot, shRNA, Negative Control, Histone Deacetylase Assay

Journal: Toxicon : official journal of the International Society on Toxinology

Article Title: Functional characterization of six aspartate (D) recombinant mojastin mutants (r-Moj): a second aspartate amino acid carboxyl to the RGD in r-Moj-D_ peptides is not sufficient to induce apoptosis of SK-Mel-28 cells

doi: 10.1016/j.toxicon.2016.04.038

Figure Lengend Snippet: αvβ3 integrin cell surface expression on untransduced, scrambled shRNA, and αv stable knocked down SK-Mel-28 cells. p<0.001 (***). αvβ5 integrin expression was not examined since we demonstrated that SK-Mel-28 cells do not express the β5 subunit (Seoane et al. 2010).

Article Snippet: Inhibition of integrin αv expression was performed in SK-Mel-28 cells using integrin αv shRNA (h) lentiviral particles (Santa Cruz Biotech, sc-29373-v).

Techniques: Expressing, shRNA

Journal: Toxicon : official journal of the International Society on Toxinology

Article Title: Functional characterization of six aspartate (D) recombinant mojastin mutants (r-Moj): a second aspartate amino acid carboxyl to the RGD in r-Moj-D_ peptides is not sufficient to induce apoptosis of SK-Mel-28 cells

doi: 10.1016/j.toxicon.2016.04.038

Figure Lengend Snippet: r-Moj-DL, r-Moj-DM, and r-Moj-DN peptides induced apoptosis of SK-Mel-28 cells by binding to the αv integrin. p=0.05 (*), p=0.01 (**), p<0.001 (***).

Article Snippet: Inhibition of integrin αv expression was performed in SK-Mel-28 cells using integrin αv shRNA (h) lentiviral particles (Santa Cruz Biotech, sc-29373-v).

Techniques: Binding Assay

Journal: Toxicon : official journal of the International Society on Toxinology

Article Title: Functional characterization of six aspartate (D) recombinant mojastin mutants (r-Moj): a second aspartate amino acid carboxyl to the RGD in r-Moj-D_ peptides is not sufficient to induce apoptosis of SK-Mel-28 cells

doi: 10.1016/j.toxicon.2016.04.038

Figure Lengend Snippet: All r-Moj-D_ mutant peptides inhibited proliferation of SK-Mel-28 cells by binding to the αv integrin. The scrambled shRNA control treated cells are not shown, since these cells grew at much slower rate than untransduced or αv knocked down cells.

Article Snippet: Inhibition of integrin αv expression was performed in SK-Mel-28 cells using integrin αv shRNA (h) lentiviral particles (Santa Cruz Biotech, sc-29373-v).

Techniques: Mutagenesis, Binding Assay, shRNA, Control

Journal: Nature Communications

Article Title: Integrin-α V -mediated activation of TGF-β regulates anti-tumour CD8 T cell immunity and response to PD-1 blockade

doi: 10.1038/s41467-021-25322-y

Figure Lengend Snippet: a Representative IHC images of tumour samples from patients with low and high α V expression in tumour cells. Objective: 20×. b Kaplan−Meier curve of OS for stage I treatment-naïve lung cancer patients according to the α V expression by IHC analysis of FFPE tumours. c Kaplan−Meier curve of PFS of PD-1 blockade-treated patients with tumours harbouring low and high expression of α V integrin. d Percentages of anti-PD-(L)1-treated patients displaying α V high tumours among long-responders (LR: PFS > 6 months and OS > 12 months) or fast progressors (FP: defined by “early death” occurring within 12 weeks of treatment initiation). e Representative digital mark-up image of fluorescent IHC of CD8 (green), cytokeratin (turquoise), and dapi (blue) staining in α V low and α V high tumour sections. d = CD8 + cell density. Left, the density of CD8 + TIL in α V low and α V high tumours. The numbers of tumours in each group are indicated (* p = 0.046). Scale bar, 2 cm. f Representative digital mark-up image of CD8 + CD103 neg (green), CD8 + CD103 + (orange), CD8 - CD103 + (red), cytokeratin (turquoise) and dapi (blue) staining in α V low and α V high tumour sections. d = CD8 + CD103 + cell density. Left, the density of CD8 + CD103 + (* p = 0.016) and CD8 + CD103 neg ( p = 0.120) cells in tumour regions of α V low and α V high tumours. Scale bar, 2 cm. Each symbol represents an individual cell type from tumour samples; horizontal lines correspond to mean ± standard error of the mean (SEM) ( e , f ). Data were calculated with the log-rank test ( b , c ) and Welch’s two-sided t -test ( e , f ). Source data are provided as a Source Data file.

Article Snippet: The cells were double transfected with integrin α V CRISPR-Cas9 KO plasmid (Santa Cruz Biotechnology, sc-400506) and integrin α V HDR plasmid (Santa Cruz Biotechnology, sc-400506-HDR).

Techniques: Expressing, Staining

Journal: Nature Communications

Article Title: Integrin-α V -mediated activation of TGF-β regulates anti-tumour CD8 T cell immunity and response to PD-1 blockade

doi: 10.1038/s41467-021-25322-y

Figure Lengend Snippet: a Representative flow cytometry plots (bi-exponential scale) of α V expression in EpCAM + E-cadherin + and EpCAM neg E-cadherin neg cells from a lung tumour. Right, percentage of α V expression in EpCAM + E-cadherin + and EpCAM neg E-cadherin neg cells ( n = 18, *** p = 0.0002). b Representative flow cytometry plots of β 6 subunit expression in EpCAM + E-cadherin + α V + and EpCAM neg E-cadherin neg α V + cells from a tumour sample. Right, expression of β 6 integrin in EpCAM + E-cadherin + α V + and EpCAM neg E-cadherin neg α V + cells ( n = 16), * p = 0.013. c Surface expression of α V , β 6 , and β 8 subunits in the IGR-B2 cell line. d Concentration of total TGF-β in CM from IGR-B2, IGR-B2T, and IGR-B2T-KO cells measured by ELISA (*** p = 0.0004). Results are presented as mean ± SEM of six independent experiments. Right, relative luciferase activity in the Mu.1LV cell line transfected with (CAGA)9-Lux reporter plasmid and treated with CM from IGR-B2, IGR-B2T, and IGR-B2T-KO cells, normalized to luciferase activity in Mu.1LV cell treated with CM from IGR-B2. Results are presented as mean ± SEM of six independent experiments (* p = 0.011, **** p < 0.0001). e Expression of α V integrin on IGR-B2T and IGR-B2T-KO cells. An isotype control was included. f Representative photos of the morphology of IGR-B2T and IGR-B2T-KO cells by phase-contrast light microscope from one experiment out of five. Objective: 20×. Each symbol represents the individual cell type from tumour samples ( a , b ); horizontal lines correspond to mean ± SEM ( a , b , d ). Data were calculated with paired Student t -tests ( a , b ) and one-way ANOVA with Tukey’s correction ( d ). ns: non-significant. Source data are provided as a Source Data file.

Article Snippet: The cells were double transfected with integrin α V CRISPR-Cas9 KO plasmid (Santa Cruz Biotechnology, sc-400506) and integrin α V HDR plasmid (Santa Cruz Biotechnology, sc-400506-HDR).

Techniques: Flow Cytometry, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Control, Light Microscopy

Journal: Toxicon : official journal of the International Society on Toxinology

Article Title: Functional characterization of six aspartate (D) recombinant mojastin mutants (r-Moj): a second aspartate amino acid carboxyl to the RGD in r-Moj-D_ peptides is not sufficient to induce apoptosis of SK-Mel-28 cells

doi: 10.1016/j.toxicon.2016.04.038

Figure Lengend Snippet: αvβ3 integrin cell surface expression on untransduced, scrambled shRNA, and αv stable knocked down SK-Mel-28 cells. p<0.001 (***). αvβ5 integrin expression was not examined since we demonstrated that SK-Mel-28 cells do not express the β5 subunit (Seoane et al. 2010).

Article Snippet: Integrin αv gene expression knockdown Inhibition of integrin αv expression was performed in SK-Mel-28 cells using integrin αv shRNA (h) lentiviral particles (Santa Cruz Biotech, sc-29373-v).

Techniques: Expressing, shRNA

Journal: Toxicon : official journal of the International Society on Toxinology

Article Title: Functional characterization of six aspartate (D) recombinant mojastin mutants (r-Moj): a second aspartate amino acid carboxyl to the RGD in r-Moj-D_ peptides is not sufficient to induce apoptosis of SK-Mel-28 cells

doi: 10.1016/j.toxicon.2016.04.038

Figure Lengend Snippet: r-Moj-DL, r-Moj-DM, and r-Moj-DN peptides induced apoptosis of SK-Mel-28 cells by binding to the αv integrin. p=0.05 (*), p=0.01 (**), p<0.001 (***).

Article Snippet: Integrin αv gene expression knockdown Inhibition of integrin αv expression was performed in SK-Mel-28 cells using integrin αv shRNA (h) lentiviral particles (Santa Cruz Biotech, sc-29373-v).

Techniques: Binding Assay

Journal: Toxicon : official journal of the International Society on Toxinology

Article Title: Functional characterization of six aspartate (D) recombinant mojastin mutants (r-Moj): a second aspartate amino acid carboxyl to the RGD in r-Moj-D_ peptides is not sufficient to induce apoptosis of SK-Mel-28 cells

doi: 10.1016/j.toxicon.2016.04.038

Figure Lengend Snippet: All r-Moj-D_ mutant peptides inhibited proliferation of SK-Mel-28 cells by binding to the αv integrin. The scrambled shRNA control treated cells are not shown, since these cells grew at much slower rate than untransduced or αv knocked down cells.

Article Snippet: Integrin αv gene expression knockdown Inhibition of integrin αv expression was performed in SK-Mel-28 cells using integrin αv shRNA (h) lentiviral particles (Santa Cruz Biotech, sc-29373-v).

Techniques: Mutagenesis, Binding Assay, shRNA